Human factor VIII:C (FVIII) is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A, a major source of hemorrhagic morbidity and mortality in affected males. Traditionally, hemophiliacs were treated with transfusions of whole blood. More recently, treatment has been with preparations of FVIII concentrates derived from human plasma. However, the use of plasma-derived product exposes hemophiliac patients to the possible risk of virus-transmissible diseases such as hepatitis and AIDS. Costly purification schemes to reduce this risk increase treatment costs. With increase in costs and limited availability of plasma-derived FVIII, patients are treated episodically on a demand basis rather than prophylactically. Recombinantly produced FVIII has substantial advantages over plasma-derived FVIII in terms of purity and safety, as well as increased availability and accordingly, much research effort has been directed towards the development of recombinantly produced FVIII. Due to the labile nature of FVIII, especially following its activation, large and repeated doses of protein whether plasma or recombinantly-derived, must be administered to achieve a therapeutic benefit. However, the amount of FVIII protein the patient is exposed to has been correlated with the development of antibodies which inhibit its activity. In light of this known immunogenicity, one of the goals in developing new recombinant forms of FVIII for use as a therapeutic agent is the development of products that reduce or eliminate such an immune response. FVIII functions in the intrinsic pathway of blood coagulation as a cofactor to accelerate the activation of factor X by factor IXa, a reaction that occurs on a negatively charged phospholipid surface in the presence of calcium ions.
The FVIII molecule is divided into 6 structural domains: a triplicated A domain (A1, A2, A3), a carbohydrate-rich and dispensable central domain (B-domain), and a duplicated C domain (C1, C2) (see FIG. 5). FVIII is secreted into plasma as a heterodimer of a heavy chain (domains A1-A2-B) and a light chain (domains A3-C1-C2) associated through a noncovalent divalent metal ion linkage between the A1- and A3-domains. In plasma, FVIII is stabilized by binding to von Willebrand factor. More specifically, the FVIII light chain is bound by noncovalent interactions to a primary binding site in the amino terminus of von Willebrand factor. Upon proteolytic activation by thrombin, FVIII is activated to a heterotrimer of 2 heavy chain fragments (A1, a 50 kDa fragment, and A2, a 43 kDa fragment) and the light chain (A3-C1-C2, a 73 kDa chain). The active form of FVIII (FVIIIa) thus consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit associated with the A1 domain through an ion association. This FVIIIa heterotrimer is unstable and subject to rapid inactivation through dissociation of the A2 subunit under physiological conditions. The FVIII molecule contains 25 consensus sequences (Asn-Xxx-Thr/Ser) that allow N-linked glycosylation, of which 20 have been shown to be glycosylated (1).
FVIII protein may be functionally defined as a factor capable of supplementing the coagulation defect in plasma derived from patients affected by haemophilia A. In order to allow the treatment of haemophilia A, FVIII has been purified from human or porcine plasma and more recently produced by recombinant DNA technologies. U.S. Pat. No. 4,965,199 discloses, for example, methods developed for the recombinant production of therapeutic quantities of FVIII in mammalian host cells. Human FVIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has also been reported and, more recently, the efficacy of B-domain deleted FVIII has been demonstrated in clinical trials (U.S. Pat. No. 4,868,112, ref 2).
Commercially available therapeutic FVIII products include plasma derived FVIII (pdFVIII) and recombinant FVIII (rFVIII) products, such as the full-length rFVIII (Kogenate® Bayer, Advate® Baxter, Helixate® CSL-Behring) and a B-domain deleted rFVIII (Refacto® Wyeth).
However, despite the availability of therapeutic grade FVIII, the need for FVIII analogues with enhanced properties remains high. Indeed, treatment of hemophilia A patients with therapeutic FVIII (pdFVIII or rFVIII) results, in 15 to 30% of the cases, in the emergence of anti-FVIII antibodies (inhibitors) which neutralize the pro-coagulant activity of the therapeutically administered FVIII (3,4). The occurrence of inhibitors is considered to reflect an allogeneic immune response to the repeated administration of an exogeneous FVIII protein. Some haemophiliacs are extremely sensitive to exogenous recombinant factor VIII and develop anti-factor VIII antibodies limiting the effectiveness of their treatment. Therefore, the development of FVIII inhibitors represents both a major medical hurdle and a critical societal concern since patients producing FVIII inhibitors become resistant to conventional replacement therapy. FVIII inhibitor occurrence not only results in a 3 folds increase of the treatment costs (5), but it also dramatically affects the quality of life of the patients, increasing morbidity and mortality. In this regard, it is highly desired to provide FVIII with reduced or absent potential to induce an immune response in the human subject. In addition, it is highly desired to provide FVIII with an increased circulation time within the human subject that would be of particular benefit in the chronic and recurring disease setting such as is the case hemophilia A.
The first step of the FVIII-directed specific immune response was shown to consist in the endocytosis of FVIII by Antigen Presenting Cells (APCs). Dendritic cells (DCs) have been suggested to be the most potent APC for priming of naïve T cells and initiation of the corresponding antigen-specific immune response (6,7). Antigen endocytosis by DCs is generally performed by macropinocytosis or by receptor-mediated endocytosis. Indeed, the DC surface presents a myriad of endocytic receptors most of which are dependent on the presence of bivalent ions, mainly calcium. Many endocytic receptors, by virtue of their exposed carbohydrate recognition domains (CRDs), are specific for sugar residues present on the antigens (8), and are referred to as C type lectin receptors (CLRs). Mannose residues on an antigen can thus be recognized by a series of mannose sensitive CLRs on DC surface, that include the mannose receptor (MR, CD206), dendritic cell specific ICAM3 grabbing nonintegrin (DC-SIGN, CD209), dectin, DEC-205 (CD205). The polycarbohydrate mannan has been shown to be a ligand for these mannose sensitive CLRs especially for MR and DC-SIGN (9-11). DC-SIGN molecule on DCs fixes the ICAM-3 on T-cells. This specific interaction seems to play a major role in the initiation of the immunological synapse between DCs and T-cells. The activation of lymphocytes might therefore be inhibited with a blocking antibody anti-DC-SIGN.
Several treatments were shown to reduce the consequences of FVIII immune response. For example treatment consisting in the use of desmopressin (a synthetic hormone which stimulate the production of FVIII), coagulation promoter agents (for example prothrombin-complex concentrate or activated prothrombin-complex concentrate), recombinant factor VIIa or perfusion of FVIII in order to induce a tolerance.
A recent method, consisting in the use of anti-idiotypic antibodies, which interact with the variable region of other antibodies, was developed to neutralize the inhibitor antibodies (12). Thus, a IgG4kappa monoclonal human antibody directed against an anti-FVIII C1 domain was isolated, which blocks the cofactor activity of FVIII and its linkage to von Willebrand factor (vWF) (13). Similarly, a human monoclonal antibody anti-FVIII C2 domain, BO2C11 (IgG4kappa) was isolated (14), which inhibits the linkage of FVIII to vWF and phospholipids. This antibody therefore inhibits completely the procoagulant activity of native and activated FVIII. An other example of monoclonal antibody is the BOIIB2, directed against FVIII A2 domain, which blocks 99% of the FVIII activity. However, the FVIII-induced immune response is a polyclonal response, and a treatment consisting in the use of anti-idiotypic antibodies directed against anti-FVIII antibodies could only partially neutralize the FVIII immune response.
The applicant has recently demonstrated that mannose-ending glycosylations on FVIII mediate the internalization of FVIII by immature human dendritic cells (DCs). These results demonstrate that blocking of the interaction between mannosylated sugars located on FVIII and the DCs mannose receptors reduces the internalization of FVIII and the further presentation to FVIII-specific T cells. Reduction of FVIII immunogenicity can thus be achieved by reducing its ability to interact with mannose-sensitive receptors.
The applicant has moreover surprisingly found that the ability of a modified FVIII wherein one or more amino acid selected from asparagin 239 (Asn239) and asparagin 2118 (Asn2118) has/have been substituted or deleted, to activate T cells when presented by DCs is substantially reduced or abolished, leading to the opportunity to provide non-immunogenic or less-immunogenic therapeutic FVIII to patients.